[go: up one dir, main page]

WO2001068681A2 - Proteines de canal ionique modifiees - Google Patents

Proteines de canal ionique modifiees Download PDF

Info

Publication number
WO2001068681A2
WO2001068681A2 PCT/GB2001/001108 GB0101108W WO0168681A2 WO 2001068681 A2 WO2001068681 A2 WO 2001068681A2 GB 0101108 W GB0101108 W GB 0101108W WO 0168681 A2 WO0168681 A2 WO 0168681A2
Authority
WO
WIPO (PCT)
Prior art keywords
ion channel
protein
gating agent
altered
gating
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2001/001108
Other languages
English (en)
Other versions
WO2001068681A3 (fr
Inventor
Alan John Aitken
Mao Xing Chen
Zoe Marie Gladwell
Andrew Jonathan Powell
Simon Nicholas Tate
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Glaxo Group Ltd
Original Assignee
Glaxo Group Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Glaxo Group Ltd filed Critical Glaxo Group Ltd
Priority to AU40822/01A priority Critical patent/AU4082201A/en
Publication of WO2001068681A2 publication Critical patent/WO2001068681A2/fr
Publication of WO2001068681A3 publication Critical patent/WO2001068681A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention concerns altered ion channel proteins.
  • the present invention concerns altered ion channel proteins having acquired sensitivity to a gating agent, that is an agent that activates the ion channel protein (or slows or otherwise inhibits the rate of inactivation), thereby permitting the flow of ions via the channel into and/or out of the host cell expressing the altered ion channel protein.
  • a gating agent that is an agent that activates the ion channel protein (or slows or otherwise inhibits the rate of inactivation), thereby permitting the flow of ions via the channel into and/or out of the host cell expressing the altered ion channel protein.
  • Voltage-gated sodium ion channels are responsible for the rising phase of the action potential and as such, play a key role in mediating electrical activity in excitable tissues.
  • the sodium ion channel is activated in response to depolarisation of the membrane. This causes a voltage-dependent conformational change in the ion channel, from a resting, closed conformation to an open, active conformation, the result of which increases the membrane permeability to sodium ions (1, 2).
  • the activation of the ion channels is usually transient (10-50ms) since the ion channels rapidly inactivate (i.e. switch to a confirmation which is unable to conduct sodium ions) during a sustained membrane depolarisation. This rapid activation and inactivation of sodium conductance causes the rapid rising phase of the action potential in excitable cells.
  • Noltage-gated sodium ion channels comprise a multisubunit complex consisting of a large (230-270kDa) highly glycosylated alpha ( ⁇ ) subunit which is usually associated with one or two of the smaller beta ( ⁇ -subunits ( ⁇ 1 , ⁇ 2 and ⁇ 3) (3, 4).
  • the alpha subunits of voltage-gated sodium ion channels form a large multigene family that has expanded over recent years and at least ten different genes have now been identified in mammals (5-13).
  • This alpha subunit consists of four homologous domains (DI-IV) each containing six potential ⁇ -helical transmembrane segments (SI-S6) which make-up the pore forming region.
  • Domains critical for the function of the channel are highly conserved throughout the family of voltage-gated sodium ion channels. These include the S4 voltage sensors, the loop between domains III and IV which is involved in the inactivation of the ion channel and the SSI and SS2 segments of the extracellular loop between transmembrane regions S5 and S6, which are responsible for the channels vestibule and ion selectivity (14-16).
  • ⁇ subunits appear to have a role in altering the kinetics of the sodium ion channel during activation and inactivation gating. Expression of the ⁇ subunits has been associated with an increase in peak current and a role in trafficking of the ⁇ subunit to the membrane (17-20).
  • the most potent blocker of voltage gated sodium ion channels is the puffer fish toxin, tetrodotoxin, (TTX). While most voltage-gated sodium ion channels are inhibited by low nanomolar concentrations of TTX, there are three ion channels which are only inhibited by micromolar concentrations of TTX. These are the major cardiac ion channel (HI or SKM2) and the sensory neurone specific ion channels, SNS/PN3 (here designated SNS1) and SNS2/NaN (3, 7, 8, 12, 13).
  • Sensory neurones of mammalian dorsal root ganglion transmit sensory information from the periphery to the central nervous system and are known to express at least three distinct kinetic types of voltage-gated sodium currents (21, 22).
  • the small diameter neurones co-express a rapidly inactivating, fast TTX sensitive current and a slowly activating and inactivating TTX resistant sodium current.
  • the larger diameter cells only express TTX sensitive sodium currents that have intermediate activation and inactivation kinetics (23, 24).
  • This electrophysiological analysis has now been supported by molecular distribution studies, which suggest that there is a dynamic expression of voltage-gated sodium ion channels in DRG neurones that can change during development, response to injury and upon exposure to inflammatory mediators (25-28).
  • the small diameter neurones are unmyelinated and are involved in the transmission of pain impulses, these neurons are the c-fibres or nociceptive neurones (29).
  • voltage-gated sodium ion channels in sensory neurones may provide a highly tractable and attractive target for the development of novel analgesic and anti-hypersensitivity agents, and thus indicated in diseases such as neuropathic pain, inflammatory pain, migraine and headache, multiple sclerosis and other demyelinating conditions (see ref. 37).
  • anaesthetic, anticonvulsant and antiarrythmic drugs each with sodium ion channel blocking activity, can produce analgesia.
  • anaesthetic, anticonvulsant and antiarrythmic drugs each with sodium ion channel blocking activity, can produce analgesia.
  • anaesthetic doses of lignocaine and bupivocaine elevate pain thresholds in man ( 8, 39).
  • the anticonvulsant agents, phenytoin, carbamazepine and the class la antiarrythmic agent mexilitene are used clinically for neuropathic pain (40-42).
  • the anticonvulsant lamotrigine is also weakly analgesic (43).
  • ⁇ -scorpion toxins bind to a common extracellular site on brain Na + ion channels thus inhibiting rapid inactivation, and that domains I and IN of the brain ⁇ a + ion channel ⁇ -subunits are implicated in ⁇ -ScTx binding (44, 45).
  • the ability of ⁇ -ScTx to prevent inactivation of the brain sodium channels provides a convenient method to gate these Na + ion channels in plate-based assays appropriate for high-throughput drug screening (46).
  • Mammalian cells expressing the brain Na + ion channel to be assayed are plated in multi-well plates and pre-incubated in a fluorescent membrane potential-sensitive dye (DiBAC(4) 3 ).
  • the transition from the open to inactivated conformation is inhibited resulting in a slight membrane depolarisation.
  • This initial depolarisation in turn causes more channels to open and not inactivate.
  • a cascade occurs that leads to a pronounced membrane depolarisation which causes a change in the fluorescence of the membrane potential-sensitive dye.
  • This fluorescence change can be detected on a fluorometric imaging plate reader (FLIPR, Molecular Devices, Sunnyvale, CA, USA; 47). If the cells are pre-incubated with agents that inhibit the Na + channel the fluorescence change upon addition of ⁇ -ScTx is reduced or completely ablated in a concentration-dependent manner. This method has allowed the activity of the brain Na + ion channels to be assayed in a plate-based system.
  • U.S.5,858,713 there is disclosed a voltage-sensitive sodium ion channel having a mutation that renders it permeable to calcium.
  • the purpose of the mutation is to allow detection of calcium flow with readily available calcium sensitive dyes.
  • the mutation of '713 is introduced into the pore itself which brings with it the risk that the ion flow detected is not an accurate representation of the in vivo situation with the unmodified ion channel.
  • modulators may be used in the preparation of pharmaceutical compositions for the treatment of a range of diseases or disorders, such as those mentioned supra, e.g. pain and hyper sensitivity.
  • an altered ion channel protein e.g. altered voltage sensitive sodium ion channel
  • ion channel protein e.g. altered voltage sensitive sodium ion channel
  • ion channel protein e.g. altered voltage sensitive sodium ion channel
  • altered ion channel protein or “altered ion channel” is intended to be understood in a structural sense as opposed to implying that for any given example, a particular process has necessarily been followed in its production.
  • an ion channel protein produced by expression of a mutagenic oligonucleotide of the invention which in turn has been produced by e.g. PCR driven amplification of an ancestry mutagenic oligonucleotide is an altered ion channel protein according to the present invention, even though the oligonucleotide it is directly produced from has not in itself undergone alteration in a process sense.
  • the altered ion channel protein has substantially increased sensitivity to a gating agent compared to the unaltered ion channel protein.
  • the increased sensitivity is at least two fold, more typically, at least 10 fold, preferably at least 100 fold.
  • the present invention provides means for conferring on an altered ion channel protein, sensitivity to gating agents in circumstances where the unaltered ion channel protein has no or little sensitivity to such agents or a particular gating agent of interest. This enables investigation of the altered ion channel protein, particularly when expressed in a host cell, and identifying modulators of the unaltered ion channel protein for use in the preparation of pharmaceutical compositions for the treatment of diseases or disorders.
  • This may be achieved by identifying within the primary amino acid sequence (or the polynucleotide e.g. DNA encoding it) of the unaltered, gating agent insensitive ion channel protein that region equivalent to a gating agent sensitive region in a family member. This may be achieved, at least in part, by e.g. sequence alignment studies.
  • the gating agent insensitive region of the unaltered ion channel protein may be changed to have a primary amino acid sequence corresponding (e.g. identical or similar/conservative change) to the gating agent sensitive region of the family member.
  • Polynucleotides encoding the altered ion channel protein may be generated by site-directed mutagenesis with mutagenic oligonucleotides being employed using a single-stranded (e.g. Ml 3 bacteriophage based techniques) or double stranded template (e.g. ChameleonTM or QuikchangeTM - StratageneTM).
  • the mutated polynucleotide is excised and inserted into a suitable vector so that the altered ion channel protein can be expressed by a host cell (although mechanically based carriers are not excluded).
  • a host cell i.e. a cassette mutation
  • splice overlapping extension PCR may be employed.
  • an altered ion channel protein according to the invention may be produced as follows: (a) identifying the binding site of a gating agent in the primary amino acid sequence of a gating agent sensitive family member;
  • step (b) aligning the sequence of step (a) with the sequence of the gating agent insensitive ion channel protein
  • step (e) incorporating the oligonucleotide of step (d) into a vector
  • step (f) introducing the vector of step (e) into a host cell
  • step (g) permitting the cell of step (f) to express said altered ion channel.
  • Determination of the gating agent binding region can be achieved by a number of routes known or apparent to those skilled in the art.
  • the binding region may be well characterised in the literature.
  • the skilled person has a number of options available.
  • synthesise of a radiolabelled gating agent and incubation of this with cells expressing said ion channel protein or alternatively with purified ion channel protein will result in a radiolabelled gating agent-ion channel protein complex which can be proteolytically digested and the fragments probed with e.g. antibodies to isolate the ion channel protein fragment - radiolabelled gating agent complex.
  • the complex can be sequenced to obtain a general picture of the binding region. Further information is then available by site-directed mutagenesis (for example alanine scanning site-directed mutagenesis) of the fragment to identify those bases critical in the binding of the gating agent. From this a comprehensive picture of the binding region can be built up.
  • site-directed mutagenesis for example alanine scanning site-directed mutagenesis
  • the term "family" is used herein to indicate a group of proteins which share substantial sequence similarities, either at the level of the primary sequence of the proteins themselves or at the level of the DNA encoding them. The sequence similarities may extend over the entire protein/gene or may be limited to particular regions or domains.
  • Similarities may be based on nucleotide/amino acid sequence identity as well as similarity (for example, those skilled in the art recognise certain amino acids as similar and identify differences based on switches of similar amino acids as conservative changes).
  • Some members of a protein family may be related in the sense that they share a common evolutionary ancestry. The members of a protein family do not necessarily share the same biochemical properties or functions though their similarities are usually reflected in common functional features.
  • the criteria by which protein families are recognised are well known in the art and include computer analysis of large collections of sequences at the level of DNA and protein as well as biochemical techniques such as hybridisation analysis and enzymatic assays (see for example Pearson and Lipman (1988) PNAS USA, 85 :2444).
  • the term "family” embraces "homologue”.
  • homologue is used herein in two distinct senses. It is used sensu stricto to define the corresponding protein from a different organism (i.e. a species variant or orthologue) in which case there is a direct evolutionary relationship between the protein and its homologue.
  • the term is also used sensu lacto to define a protein which is structurally similar to a given ion channel protein. In this sense, homology is recognised on the basis of purely structural criteria by the presence of amino acid identities and/or conservative amino acid changes (as set forth by Dayhoff et al, Atlas of protein structure, vol. 5, National Biomed Dd'n, Washington D.C., 1979).
  • the unaltered ion channel protein has at least 35% identity with the gating agent sensitive family member or homologue.
  • the ion channel protein has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, more preferably, at least 90%, 95%, 96%, 97%, 98% or even 99% identity.
  • the present invention is of particular use in conferring gating agent sensitivity on fast voltage gated ion channel proteins which are insensitive or otherwise refractory to gating agents.
  • Preferred examples are sodium ion channel proteins such as the sensory neurone specific (SNS) family as disclosed in WO 99/47670 (SNS2) WO 97/01577 (here termed SNSl) and purportedly so in Jeong et al (2000; ref. 48). The reader is specifically referred to each reference.
  • SNSl as disclosed in WO 97/01577 which is insensitive to ⁇ -scorpion toxin.
  • an altered sodium ion channel protein particularly SNSl ion channel protein having acquired sensitivity to a gating agent, particularly ⁇ -scorpion toxin.
  • the altered SNS ion channel protein comprises a region corresponding to (e.g. identical or similar) to a sodium ion channel protein family member that has sensitivity to ⁇ - scorpion toxin such as the brain I, II or III sodium ion channel protein.
  • the alteration takes place in the SNS ⁇ subunit.
  • ⁇ -scorpion toxin sensitive and insensitive family members are shown in table 1 below.
  • an altered rat SNS 1 ion channel protein wherein positions 1554 to 1569 inclusively are identical to or similar to positions 1607 to 1620 inclusively in rat brain II sodium ion channel protein so as to confer gating agent sensitivity on said altered rat SNSl ion channel protein to ⁇ -scorpion toxin.
  • Host cells expressing said altered rat SNSl ion channel protein as hereinbefore described are also provided.
  • an altered human SNSl sodium ion channel protein having gating agent sensitivity particularly to scorpion toxin.
  • Positions 1552 to 1566 inclusively of said altered human SNSl sodium ion channel protein comprises a plurality of contiguous bases derived from a gating agent sensitive region of a gating agent sensitive family member such as a brain sodium ion channel (e.g. I, II or III).
  • a gating agent sensitive region meaning that region responsible for conferring gating agent sensitivity on the gating agent sensitive family member.
  • an altered mouse SNS 1 sodium ion channel protein having gating agent sensitivity particularly to ⁇ - scorpion toxin.
  • Positions 1553 to 1567 inclusively of said altered mouse SNSl sodium ion channel protein comprises a plurality of contiguous bases derived from a gating agent sensitive region of a gating agent sensitive family member such as a brain sodium ion channel.
  • an altered rat SNSl sodium ion channel protein having gating agent sensitivity particularly to scorpion toxin.
  • Positions 1554 to 1569 inclusively of said altered rat SNSl sodium ion channel protein comprises a plurality of contiguous bases derived from a gating agent sensitive region of a gating agent sensitive family member such as a brain sodium ion channel or similar thereto.
  • derived from is intended to define not only a source in the sense of it being the physical origin for the material but also to define material which has structural and/or functional characteristics which correspond to those of material which does originate from the source. Thus a "plurality of contiguous bases derived from a gating agent sensitive region" need not necessarily have been purified from the gating agent sensitive family member.
  • the unaltered, gating agent insensitive ion channel protein is mammalian, more preferably rat (protein is SEQ.I.D.NO:l, DNA is SEQ.I.D.NO:2), mouse (protein is SEQ.I.D.NO:3, DNA is SEQ.I.D.NO:4) or human (protein is SEQ.I.D.NO:5, DNA is SEQ.I.D.NO:6) of which human is most preferred.
  • a human sodium ion channel protein such as SNSl or SNS2.
  • the alteration to the gating agent insensitive amino acid region of the unaltered ion channel protein may take the form of a single base change or a plurality of base changes (which plurality of bases maybe contiguous). Indeed, a single amino acid change may in some circumstances be insufficient for conferring gating agent sensitivity on the altered ion channel protein.
  • Fig. 1 when the primary amino acid sequences of the D-IVS3 to D-IVS4 region of voltage-gated Na + channel family members are aligned (Fig. 1), the acidic glutamic acid residue at 1613 (brain Ila) is conserved across the rat brain channels whereas in the SNS channels it is neutral (rat SNS) or basic (rat SNS2).
  • the glutamic acid residue at 1616 (brain Ila) is also highly conserved across the brain channels, whereas in rat SNS it is basic.
  • a single base change in the unaltered gating agent insensitive ion channel protein in a position equivalent to a single base in a family member that is gating agent sensitive may not be sufficient to confer acquired sensitivity on the altered ion channel protein, even if the change corresponds to the single base of the family member.
  • a plurality of changes such as a cassette mutation in a polynucleotide sequence encoding the unaltered ion channel protein.
  • alteration is achieved through the provision of polynucleotide(s) encoding the altered ion channel protein which is then inserted into a suitable vector as outlined above. It will be apparent to those skilled in the art that due to the degeneracy of the genetic code, many variations of the polynucleotide are possible. Such changes may be by way of addition, substitution, deletion, insertion of one or more polynucleotide bases.
  • region includes a single base or a plurality of bases, which plurality may or may not be contiguous. It is preferred that the number of alterations introduced into the primary amino acid sequence to confer acquired gating agent sensitivity is kept to a minimum, i.e. as few changes as are necessary. This ensures that the resulting functional character of the altered ion channel protein reflects as far as possible the unaltered ion channel protein.
  • Suitable vectors may be a plasmid, phage, minichromosome or transposon of which plasmid is particularly preferred.
  • the vector may include other regulatory elements such as a promoter, operator, activator, repressor and/or enhancer, transcription, translation, initiation and termination sequences. They may also contain sequence encoding any of various tags (e.g. to facilitate subsequent purification of the expressed protein such as affinity (e.g. His) tags.
  • the altered ion channel protein is a sodium ion channel protein, particularly SNSl
  • vectors of the present invention may also comprise polynucleotide sequences encoding the unaltered ⁇ subunit of the sodium channel protein.
  • the present invention specifically contemplates expression of an altered ⁇ subunit of a sodium ion channel particularly SNSl, either alone or together with one or more ⁇ sodium ion channel subunit(s) to form an altered sodium ion channel protein.
  • vectors which comprise a regulatory element or elements operably linked to the mutagenic polynucleotide (e.g. DNA) of the present invention to provide expression thereof at suitable levels (e.g. at a level which facilitates its use in drug screening assays).
  • a regulatory element or elements operably linked to the mutagenic polynucleotide (e.g. DNA) of the present invention to provide expression thereof at suitable levels (e.g. at a level which facilitates its use in drug screening assays).
  • operably linked refers to a condition in which portions of a linear polynucleotide sequence are capable of influencing the activity of other portions of the same linear sequence, for example, a ribosome binding site is operably linked to a coding sequence if it positioned so as to permit translation.
  • the vector may be inserted into the host cell according to any conventional method. It is preferred that the altered sodium ion channel has acquired sensitivity to ⁇ -scorpion toxin although the acquired sensitivity may be towards one or more of the following gating agents;
  • Veratridine batrachotoxin, aconitine, grayanotoxin, sea anemone, ⁇ scorpion toxin, brevetoxin, ciguatoxin, deltamethrin or cypermethrin. It is preferred that the altered ion channel protein is amenable to ion flow by its natural ion to better reflect the in vivo situation as opposed to the approach taken in US 5,858,713.
  • Host cells harbouring the vectors of the present invention are preferably eukaryotic , more preferably mammalian, most preferably human, e.g. a human cell such as a HEK 293 cell. Also preferred are mammalian cell lines such as neuroblastoma. For screening purposes it is preferred to use a human cell line so as to best represent the in vivo situation.
  • Vectors of the present invention maybe inserted into a host cell by way of transfection according to techniques well known or apparent to those skilled in the art. Liposomes and other mechanical carriers are not excluded.
  • Host cells expressing the altered ion channel protein may then be used in assays for identifying modulators of the unaltered ion channel protein.
  • the modulator may be an agonist, antagonist or mimetic.
  • the modulator is an antagonist.
  • a candidate agent By altering the ion channel protein so as to render it sensitive to a gating agent, those skilled in the art will recognise that in drug screening assays, particularly for an antagonist, the ability of a candidate agent to modify activation of the altered ion channel protein when in the presence of the gating agent is indicative of a potential modulator of the unaltered ion channel protein in vivo.
  • Such candidate agents may be identified and further selected and/or modified (i.e. developed) to produce a modulator of the unaltered ion channel protein in vivo (particularly for use in treating a mammalian patient such as a human).
  • Further selection and/or modification criteria may include one or more of the following considerations; Potency as a ion channel protein modulator, selectivity (towards the target unaltered ion channel protein), pharmacokinetics (e.g. ADME in animal models), in vitro electrophysiology characteristics (e.g. on dorsal sensory neurones or spinal nerve preparations), in vivo activity in appropriate animal models of disease (e.g. chronic constriction injury, model of neuropathic pain, carageenan model of inflammatory pain), chemical synthesis, stability and formulation characteristics of the candidate agent, safety and toxicology (acute and chronic) studies in animals. Development may then proceed to proof of concept studies in man before clinical trials begin.
  • Potency as a ion channel protein modulator e.g. ADME in animal models
  • in vitro electrophysiology characteristics e.g. on dorsal sensory neurones or spinal nerve preparations
  • in vivo activity in appropriate animal models of disease e.g. chronic constriction injury, model of neuropathic
  • a candidate modulator which is capable of modulating the altered SNS ion channel protein in vitro does not also modulate activation of the unaltered cardiac sodium ion channel protein in vitro is selected or vice-versa.
  • the aim of which is to minimise potential side effects in vivo as a result of unwanted or inappropriate interaction of a SNS ion channel protein modulator with the cardiac sodium ion channel and vice-versa.
  • an ion channel protein modulator particularly an antagonist of ion channel protein activation, which ion channel protein (which is preferably a sodium ion channel, such as SNS 1 or SNS2) is insensitive or otherwise refractory to known gating agents in vitro which method comprises the steps of;
  • step (d) and optionally synthesising and/or purifying said ion channel modulator of step (c).
  • Illustrative of commercially available assays that may be employed in identifying a candidate ion channel modulators include Voltage Imaging Plate Reading (VIPR, Aurora Biosciences Corporation), Fluorometric Imaging Plate Reading (FLIPR, Molecular Devices) or the method involving bioluminescence resonance energy transfer (BRET) as disclosed in WO 99/66324 and to which the reader is specifically referred.
  • VIPR Voltage Imaging Plate Reading
  • FLIPR Fluorometric Imaging Plate Reading
  • BRET bioluminescence resonance energy transfer
  • Ion channel protein modulators identified by (or identifiably by) said method are also provided.
  • Medicaments, typically pharmaceutical compositions, comprising said modulator are also provided.
  • Such pharmaceutical compositions generally comprise a therapeutically effective amount of the modulator, usually in unit dosage form together with a pharmaceutically acceptable carrier, excipient and the like as known and called for by accepted pharmaceutical practice.
  • Use of a modulator in the manufacture of a medicament for the treatment of pain and/or a hypersensitivity disease or disorder and methods of treating the same are provided.
  • an altered mammalian cardiac sodium ion channel protein having acquired sensitivity to a gating agent.
  • a polynucleotide capable of encoding an altered ion channel protein as hereinbefore described.
  • polynucleotides capable of hybridising to said encoding polynucleotides.
  • hybridisable polynucleotides hybridise under stringent conditions according to methods well known to those skilled in the art. The exact conditions employed are a matter of choice for the skilled person but stringency may be manipulated by varying temperature and salt conditions. Further guidance may be sought from standard textbooks in the art such as Sambrook J et al, Molecular Cloning: A laboratory manual, 2 nd Edtn, Cold Spring Harbour Labs.
  • Figure 1 Alignment of SNS, brain, cardiac and neuroendocrine sodium channels across the conserved ⁇ -ScTx binding site of the brain channels between domains IVS3 and IVS4. The two conserved amino acids are underlined while the extracellular end of the IVS3 domain is indicated (/).
  • Figure 2 Alignment showing the amino acid sequence substituted into rSNSl from brain II (in bold) to generate the rSNS-bll mutant (SNS mutant 1). The two conserved amino acids are underlined while the extracellular end of the IVS3 domain is indicated (/).
  • Figure 3 DNA alignments showing the single base pair changes made to generate the single and the double amino acid substitutions (SNS mutants 2 and 3 respectively).
  • Figure 6 Analysis of ⁇ -ScTx sensitivity of wild type rSNSl, human brain IIA and the three novel rSNSl mutants by patch-clamp electrophysiology.
  • Figure_7 Analysis of ⁇ -ScTx sensitive SNS mutant 1 in a FLIPR DiBAC assay.
  • the rat SNS cDNA (rSNSl) was cloned by screening a rat DRG lambda ZAP express cDNA library (constructed by GW; Clontech vector) using a colony hybridisation protocol, and by RT-PCR performed on rat DRG total RNA. This resulted in 7 fragments spanning the coding region which were then assembled in the vector, pBluescript SK + (Stratagene). This plasmid was then modified by site directed mutagenesis to correct a number of PCR derived mutations and to insert a deleted nucleotide.
  • the rat SNSl coding region was then subcloned into a pBR322-based shuttle vector pBRLT7 using the Notl and Kpnl enzyme restriction sites to give pBRLT7- rSNSl (Fig. 4b).
  • the Notl and Mlul sites were then used to transfer the coding region into an IRES mammalian expression vector, pCIN5 (GW) which was digested with Notl and Ascl (Mlul compatible) restriction enzymes, to give pCIN5-rSNSl (Fig. 4c).
  • mutant 1 (Fig.2 shows relevant amino acid changes)
  • mutant 2 (Fig.2 shows relevant amino acid changes)
  • mutant 3 (Fig.2 shows relevant amino acid changes)
  • mutant 4a The entire mutant coding region (rSNSl-BII) was excised by Hindlll and Kpnl restriction enzymes, and inserted into the BstXl-Nsil restriction sites of the IRES mammalian expression vector, pCIN5-PL (GW) by blunt end ligation.
  • the resulting construct was designated pCIN5-rSNSl-rBII and the encoded mutant protein designated rSNSl mutant 1. Constructing the point mutations in the rat SNS cDNA by site directed mutagenesis:
  • the rat SNSl coding region was sub-cloned into the plasmid pBK-CMV (Stratagene) as described previously, to give pBK-CMV-rSNSl (Fig.4a).
  • Complementary oligonucleotide primer pairs were designed which incorporated the required single and double nucleotide mutations respectively.
  • the mutagenesis was performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). For each mutagenesis reaction 50ng of pBK-CMV- rSNSl was combined with 125ng of each complementary primer, l ⁇ l of dNTP (25mM of each NTP), 5 ⁇ l of reaction buffer, ddH 2 O to a final volume of 50 ⁇ l and l ⁇ l of Pfu DNA polymerase (2.5U/ ⁇ l).
  • Dpnl restriction enzyme (lOU/ ⁇ l) was added to each amplification reaction and incubated in a water bath at 37°C for 1 hour, then l ⁇ l of each reaction was transformed into 50 ⁇ l E. coli XL2-Blue supercompetent cells.
  • the transformed bacterial cell suspension plus 450 ⁇ l of NZY+ broth was pre- incubated for 1 hour at 28°C with shaking at 225rpm, before the entire volume was plated out onto LB agar plates supplemented with 50 ⁇ g/ml of ampicillin. The plates were then incubated for 18 hours at 28°C. Plasmid DNA was purified from overnight cultures of single colonies picked from these plates using the miniprep spin column protocol (Qiagen). The mutations, designated mutant 2 and 3 for the single and double mutations respectively (Fig.3), were then verified by sequencing the plasmid DNA.
  • Mammalian cells (either H ⁇ K293, A, C, D & E or CHO, B) were stably (A, B & C) or transiently (D & E) transfected with expression constructs ( Figures 4 & 5) for the wild type or mutant sodium ion channel ⁇ -subunits indicated.
  • Whole-cell patch clamp recordings were made from these cells using standard methods (50). Briefly, cells were grown on a glass coverslip, placed into a recording chamber (0.5ml volume) and superfused with an extracellular recording solution at 2 ml min "1 .
  • the extracellular recording solution contained (in mM): 140 NaCl, 4.7 KC1, 1.2 MgCl 2 , 1 CaCl 2 , 11 glucose, 5 HEPES (pH 7.4).
  • Patch electrodes had resistances of 2 to 6M ⁇ when filled and the pipette-filling solution contained (in mM): 120 CsF, 15 NaCl, 10 Cs- EGTA (ethylene glycol-bis( ⁇ -aminoethyl ester) N,N,N',N-tetra acetic acid Cs salt), 10 HEPES. Currents were recorded at room temperature using an Axopatch 200A amplifier (Axon Instruments Inc.).
  • the expression plasmids pCIN5-rSNSlwt and pCIN5-rSNSl -mutant 1 were transfected into HEK293 cells by electroporation and stable HEK293 clonal cell lines were isolated following selection in 800 ⁇ g/ml Geneticin G418 (Life Technologies). Two cell lines that functionally expressed the SNS proteins were identified by electrophysiology (one expressing wild type rSNSl and one expressing the ⁇ -ScTx-sensitive rSNSl mutant 1) and these cell lines were used for the FLIPR assays described here.
  • the two cell lines were plated in black, clear bottomed 96 well assay plates (Costar®, Corning Inc.) at a seeding density of 5 x 10 4 cells per well and incubated for 48 hours prior to assay (37°C). Native HEK293 cells were also plated as a negative control.
  • the cells were loaded with the membrane potential sensitive dye, DiBAC(4) 3 5 ⁇ M, Molecular Probes) either in the presence or absence of 30 ⁇ g/ml ⁇ -ScTx in low sodium buffer (5mM NaCl, 140mM tetramethyl ammonium-Cl, 5mM KC1, 2mM CaCl 2 , 0.8mM HEPES, lOmM D-glucose, pH7.4, plus 0.5mM Brilliant Black dye; lOO ⁇ l per well) at 37°C for 30 minutes.
  • low sodium buffer 5mM NaCl, 140mM tetramethyl ammonium-Cl, 5mM KC1, 2mM CaCl 2 , 0.8mM HEPES, lOmM D-glucose, pH7.4, plus 0.5mM Brilliant Black dye; lOO ⁇ l per well
  • the plates were transferred to the FLIPR and the cell-loaded DiBAC fluorescence (excited at 488nm) assayed during and following addition of lOO ⁇ l of sodium add-back buffer (145mM NaCl, 5mM KC1, 2mM CaCl 2 , 0.8mM HEPES, lOmM D-glucose, pH7.4, plus 0.5mM Brilliant Black dye) to each well.
  • lOO ⁇ l of sodium add-back buffer 145mM NaCl, 5mM KC1, 2mM CaCl 2 , 0.8mM HEPES, lOmM D-glucose, pH7.4, plus 0.5mM Brilliant Black dye
  • Figures 7 A and 7B show the representative traces of changes in fluorescence in control HEK293, HEK293 rSNSl wild type (wt) and HEK293 rSNSl mutant 1 ion channel cells following 30 minutes incubation in the absence (A) or presence (B) of 30 ⁇ g/ml ⁇ -ScTx. Similar responses as those in the representative traces were obtained in duplicate wells. In the absence of ⁇ - ScTx preincubation, only a small depolarisation occurs in each of the 3 cell types upon addition of sodium. This depolarisation does not correspond to SNS ion channel expression since it occurs in the native HEK293.
  • the HEK293 rSNSl mutant 1 (the ⁇ -ScTx-sensitive rSNSl mutant) gives a significant increase in fluorescence that corresponds to membrane depolarisation indicating that the ⁇ -ScTx has gated the mutant ion channel to allowing sodium to flow through.
  • the wild type rSNSl and native HEK cells fail to give this increase in fluorescence indicating that the ⁇ -ScTx only gates the ScTx-sensitive mutant ion channel.
  • mutating ofthe rSNSl ion channel in order to change its sensitivity to a gating agent in this case ⁇ - ScTx
  • NaN a novel voltage-gated Na channel
  • Type III sodium channel mRNA is expressed in embryonic but not adult spinal sensory neurones, and is re-expressed following axotomy. J. Neurophysiol., 72, 466- 470.
  • PGE 2 modulates the tetrodotoxin-resistant sodium current in neonatal rat dorsal root ganglion neurones via the cyclic AMP-protein kinase A cascade. J. Physiol., 495, 429- 440. 31. Cardenas, C.G., Del Mar, L.P., Cooper, B.Y. & Scroggs, R.S. (1997). 5- HT receptors couple positively to tetrodotoxin-insensitive sodium channels in a subpopulation of capsaicin-sensitive rat sensory neurones. J. Neurosci., 17, 7181-7189.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des protéines de canal ionique modifiées possédant une sensibilité acquise à des agents activateurs. L'invention porte sur les canaux ioniques réfractaires auxdits agents activateurs. Les protéines de canal ionique modifiées de l'invention sont utiles dans l'identification de modulateurs du canal ionique non modifié. L'invention concerne également des cellules hôte, des vecteurs et des analyses de criblage.
PCT/GB2001/001108 2000-03-14 2001-03-14 Proteines de canal ionique modifiees Ceased WO2001068681A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU40822/01A AU4082201A (en) 2000-03-14 2001-03-14 Altered ion channel proteins

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0006024A GB0006024D0 (en) 2000-03-14 2000-03-14 Altered ion channel proteins
GB0006024.4 2000-03-14

Publications (2)

Publication Number Publication Date
WO2001068681A2 true WO2001068681A2 (fr) 2001-09-20
WO2001068681A3 WO2001068681A3 (fr) 2002-01-24

Family

ID=9887523

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2001/001108 Ceased WO2001068681A2 (fr) 2000-03-14 2001-03-14 Proteines de canal ionique modifiees

Country Status (3)

Country Link
AU (1) AU4082201A (fr)
GB (1) GB0006024D0 (fr)
WO (1) WO2001068681A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004087956A3 (fr) * 2003-04-02 2005-03-03 Ionix Pharmaceuticals Ltd Lignees cellulaires

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9513180D0 (en) * 1995-06-28 1995-08-30 Univ London Ion channel
GB9805793D0 (en) * 1998-03-18 1998-05-13 Glaxo Group Ltd Sodium ion channels

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004087956A3 (fr) * 2003-04-02 2005-03-03 Ionix Pharmaceuticals Ltd Lignees cellulaires

Also Published As

Publication number Publication date
GB0006024D0 (en) 2000-05-03
WO2001068681A3 (fr) 2002-01-24
AU4082201A (en) 2001-09-24

Similar Documents

Publication Publication Date Title
US7790845B2 (en) Bacterial ion channel
Rappas et al. Comparison of orexin 1 and orexin 2 ligand binding modes using X-ray crystallography and computational analysis
Kalia et al. From foe to friend: using animal toxins to investigate ion channel function
Bende et al. A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a
JP6324952B2 (ja) αＯ−スーパーファミリーコノトキシンペプチド、これらの医薬組成物及びこれらの使用
US20040171114A1 (en) Isolation and use of ryanodine receptors
AU2010201829A1 (en) Endogenous and non-endogenous versions of human G protein-coupled receptors
JPH08509607A (ja) ヒトｎ−メチル−ｄ−アスパラギン酸受容体サブユニット、これをコードする核酸およびその用途
JP2004526441A (ja) 内因性および非内因性型ヒトｇタンパク質共役受容体
Cohen et al. Voltage-dependent sodium channels
US20060121460A1 (en) Toll-like receptor 11
Senatore et al. Transient and big are key features of an invertebrate T-type channel (LCav3) from the central nervous system of Lymnaea stagnalis
Zhu et al. Discovery, characterization, and engineering of LvIC, an α4/4-conotoxin that selectively blocks rat α6/α3β4 nicotinic acetylcholine receptors
US7157243B1 (en) DNA encoding mammalian T-type calcium channels
CA2489405A1 (fr) Procede de criblage d'un agent pour accroitre la teneur en insuline
JP2002504492A (ja) Ｇタンパク質共役型受容体Ｆｉｓｈｂｏｙ
US7972813B2 (en) Tetrodotoxin-resistant sodium channel alpha subunit
WO2001068681A2 (fr) Proteines de canal ionique modifiees
AU780460B2 (en) Nicotinic acetylcholine receptor alpha10 subunit
US6660496B1 (en) Nucleic acid molecules encoding a KOR3 kappa opioid receptor and the methods of producing the encoded receptor
JPH0625294A (ja) ヒトグルタメート受容体タンパク質および関連のｄｎａ化合物
RU2190663C2 (ru) Белок атр-чувствительного калиевого канала и кодирующий его дезоксирибонуклеотид
JPH10508751A (ja) ｐａｒａカチオンチャンネルモジュレーターの同定方法
US20090176243A1 (en) Novel monkey GPR103 and monkey QRFP and method of evaluating compound by using GPR103
JP4797508B2 (ja) 新規グルタミン酸受容体とその利用

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP